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How Do We Study the Dynamic Structure of Unstructured Proteins: A Case Study on Nopp140 as an Example of a Large, Intrinsically Disordered Protein Jung-Hyun Na 1 , Won-Kyu Lee 2 and Yeon Gyu Yu 1, * 1 2

*

Department of Chemistry, Kookmin University 77, Jeongneung-Ro, Seongbuk-gu, Seoul 02707, Korea; [email protected] New Drug Development Center, Osong Medical Innovation Foundation, Osong Sengmyung-Ro 123, Osong-eup, Heungdeok-gu, Cheongju-si, Chungbuk 28160, Korea; [email protected] Correspondence: [email protected]; Tel.: +82-2-910-5421

Received: 4 January 2018; Accepted: 23 January 2018; Published: 27 January 2018

Abstract: Intrinsically disordered proteins (IDPs) represent approximately 30% of the human genome and play key roles in cell proliferation and cellular signaling by modulating the function of target proteins via protein–protein interactions. In addition, IDPs are involved in various human disorders, such as cancer, neurodegenerative diseases, and amyloidosis. To understand the underlying molecular mechanism of IDPs, it is important to study their structural features during their interactions with target proteins. However, conventional biochemical and biophysical methods for analyzing proteins, such as X-ray crystallography, have difficulty in characterizing the features of IDPs because they lack an ordered three-dimensional structure. Here, we present biochemical and biophysical studies on nucleolar phosphoprotein 140 (Nopp140), which mostly consists of disordered regions, during its interaction with casein kinase 2 (CK2), which plays a central role in cell growth. Surface plasmon resonance and electron paramagnetic resonance studies were performed to characterize the interaction between Nopp140 and CK2. A single-molecule fluorescence resonance energy transfer study revealed conformational change in Nopp140 during its interaction with CK2. These studies on Nopp140 can provide a good model system for understanding the molecular function of IDPs. Keywords: intrinsically disordered protein (IDP); nucleolar phosphoprotein 140 (Nopp140); conformational study; casein kinase 2 (CK2)

1. Introduction A well-defined tertiary structure was considered until recently to be the determining factor for biological and biochemical function of proteins. Active sites of enzymes or interaction domains of signaling proteins usually consist of amino acid residues in their tertiary structure. However, stretches of amino acids lacking a stable tertiary structure are often found in linker regions between well-folded domains. These flexible linker regions provide relative motional freedom between the connected domains, as observed in the linker region between the Fab and Fc domains of immunoglobulin G. On the other hand, long stretches of unstructured regions (usually >40 amino acids) are frequently found in various proteins, and these disordered regions are considered to be an intrinsic property of the protein. Proteins containing such disordered regions are called intrinsically disordered proteins (IDPs) or intrinsically unstructured proteins [1]. Genome-wide analysis suggests that approximately 25% of the proteins in eukaryotes contain disordered regions with >50 amino acids [2]. Even in archaea and bacteria, a significant portion of proteins contain disordered regions [2]. In humans, IDPs are found in various protein families, such as transcription Int. J. Mol. Sci. 2018, 19, 381; doi:10.3390/ijms19020381

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IDPs are found in various protein families, such as transcription factors, amyloid proteins, ribosomal proteins, and ribonucleoprotein complexes. Among them, IDPs, such as nucleolar phosphoprotein 140 factors, amyloid proteins, ribosomal proteins, and ribonucleoprotein complexes. Among them, IDPs, (Nopp140) and Treacle [3], are distinctive IDPs which consist of >80% intrinsically disordered regions such as nucleolar phosphoprotein 140 (Nopp140) and Treacle [3], are distinctive IDPs which consist of (IDRs) (Figure 1). The length of IDRs in these proteins is often >200–300 aa, and these sequences are >80% intrinsically disordered regions (IDRs) (Figure 1). The length of IDRs in these proteins is often characterized as having a high percentage of disorder-promoting residues [4] (Table 1). Although these >200–300 aa, and these sequences are characterized as having a high percentage of disorder-promoting IDPs lack a well-folded domain structure, they have several motifs that can serve as interaction sites residues [4] (Table 1). Although these IDPs lack a well-folded domain structure, they have several with other biomolecules, such as DNA, RNA, or protein [5]. Previous studies have indicated that motifs that can serve as interaction sites with other biomolecules, such as DNA, RNA, or protein [5]. these interactions associated biological disorders [6–8]. Previous studies are have indicatedwith that numerous these interactions are functions associated and withhuman numerous biological During interactions with target[6–8]. proteins, particularly IDRs, may undergo conformational functions and human disorders DuringIDPs, interactions with target proteins, IDPs, particularly IDRs, changes. It is challenging to characterize structural features of IDPs during interaction because there may undergo conformational changes. It is challenging to characterize structural features of IDPs areduring only ainteraction limited number experimental techniques which are useful techniques in the structural analysis because of there are only a limited number of experimental which are useful of IDPs. In this review, we summarize recent experimental methods targeting IDPs, particularly those in the structural analysis of IDPs. In this review, we summarize recent experimental methods targeting which consist of IDRs, further present Nopp140 as anpresent example for structural analysis IDPs,mainly particularly those whichand mainly consist of IDRs, and further Nopp140 as an example using experimental methods, such as single-molecule resonance energy transfer for structural analysis using experimental methods, such asfluorescence single-molecule fluorescence resonance energy transfer (smFRET), electronresonance paramagnetic resonance nuclear magnetic(NMR), resonance (smFRET), electron paramagnetic (EPR), nuclear(EPR), magnetic resonance and(NMR), circular and circular dichroism (CD). dichroism (CD).

Figure 1. 1. Computational in nucleolar nucleolarphosphoprotein phosphoprotein140 140(Nopp140) (Nopp140) Figure Computationalprediction predictionof ofdisordered disordered regions regions in and Treacle iupred.enzim.hu/) [9]. If the the residue residuevalue valueofofthese these proteins and Treacleusing usingIUPred IUPredserver server (http:// (http://iupred.enzim.hu/) [9]. If proteins exceeds a threshold (dotted line), the residue is considered disordered. exceeds a threshold (dotted line), the residue is considered disordered. Table 1. The percent ofofdisordered-promoting (P,E, E,S,S,Q, Q,and andK)K)ofofNopp140 Nopp140 and Treacle. Table 1. The percent disordered-promoting residues residues (P, and Treacle. Disordered protein Disordered protein % of disordered-promoting residues

% of disordered-promoting residues

Nopp140 54.4

Nopp140 54.4

Nucleolar phosphoprotein 140 (Nopp140)

Treacle Treacle 49.1 49.1

Nucleolar phosphoprotein 140 (Nopp140).

2. Molecular Mechanisms of IDPs: Regulation of the Target Protein by Specific Interaction 2. Molecular Mechanisms of IDPs: Regulation of the Target Protein by Specific Interaction

Most IDPs execute their function via molecular recognition of target molecules, except for a few Most execute their function molecular recognition target molecules, except foror a few IDPs that actIDPs via entropic function [10].via Target molecules of IDPsofare nucleic acids, proteins, small IDPs that act via entropic function [10]. Target molecules of IDPs are nucleic acids, proteins, or small molecules. The function of the target molecules is altered after interaction with IDPs. On binding, molecules. molecules altered after interaction IDPs. On binding,example IDPs IDPs can alterThe thefunction activityofofthe thetarget target moleculeisin numerous ways. Onewith well-characterized can alter the activity of the target molecule in numerous ways. One well-characterized example is p53. is p53. IDR in the N-terminus of p53 (TAD of p53) interacts with p53-specific E3 ubiquitin ligase IDR in the N-terminus of p53 (TAD of p53) interacts with p53-specific E3 ubiquitin ligase Mdm2. Upon Mdm2. Upon binding, Mdm2 continuously ubiquitinates p53 and mediates its degradation by binding, Mdm2 continuously ubiquitinates p53 and mediates its degradation by proteasomes [11]. proteasomes [11]. In the p53 TAD, a 15-aa peptide serves as the binding site for the hydrophobic In the p53 TAD, a 15-aa peptide serves as the binding site for the hydrophobic pocket at the N-terminus pocket at the N-terminus of Mdm2. Structural analysis of the interaction between Mdm2 and the of Mdm2. Structural analysis of the interaction between Mdm2 and the binding peptide of p53 indicates binding peptide of p53 indicates that the binding peptide of p53 adopts a helical structure from a that the binding peptide of p53 adopts a helical structure from a more disordered conformation [12]. more disordered conformation IDPsinoften with several subunits multi-subunit IDPs often interact with several[12]. subunits largeinteract multi-subunit complexes, such in as large ribonucleoprotein complexes, such asribosomes, ribonucleoprotein particlesor (RNPs), ribosomes, the cytoskeleton, or In transcription particles (RNPs), the cytoskeleton, transcription pre-initiation complexes. this case, pre-initiation complexes. In this case, IDPs serve as assemblers. U1-70k is IDP in which the disordered regions represent 60% of the whole protein. Structural analysis of U1 snRNP complexes showed that U1-70k wraps around the core domain of U1 snRNP and interacts with several components of the U1

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IDPs serve as assemblers. U1-70k is IDP in which the disordered regions represent 60% of the whole protein. Structural analysis of U1 snRNP complexes showed that U1-70k wraps around the core domain of U1 snRNP and interacts with several components of the U1 snRNP particle [13]. This configuration implies that IDPs can stabilize the interaction between subunits in multi-subunit complexes. The structure of this complex demonstrates how IDP can interact with several protein components, stabilizing the complex protein structure as well as its own disordered regions. 3. Conformational Study of IDPs Using Experimental Techniques Because IDPs are associated with various biological functions related to human disorders [6–8], it is important to understand their physiological properties. A conformational study is a common approach for understanding underlying mechanisms of protein function. Compared with globular proteins, it is difficult to study structural properties of IDPs using conventional methods, such as X-ray crystallography, because of the inherent structural flexibility of IDPs. For these reasons, bioinformatical approaches, such as using amino acid sequences to deduce the properties of IDPs, are preferred for their identification and structural characterization [5]. Bioinformatical analysis can predict disordered regions and specific regions related to protein–protein interaction [5]. For detailed characterization of properties, such as structural dynamics in solution, however, a combination of bioinformatical and experimental methods are required [14]. Here, we introduce some of the preferred experimental methods for understanding structural features of IDPs. 3.1. NMR Spectroscopy NMR spectroscopy is a structural biology technique which is suitable as a complement to X-ray crystallography. A triple-resonance experiment (13 C, 15 N, and 1 H), which detects sequential connectivity between neighboring residues, is typically used. For this experiment, it is necessary to purify isotope-labeled proteins (with 13 C and/or 15 N) expressed in culture media containing 13 C glucose, 13 C acetate, and/or 15 NH4 Cl. The NMR signal of protein sometimes display severe spectral overlap [15]. To overcome this problem, enhanced NMR technique and spectral signal analysis by sparse multi-dimensional Fourier Transform processing are used [15]. NMR may be suited for the conformational characterization of IDPs. The conformation of IDPs is sensitive to environmental changes (e.g., pH, temperature, ionic strength, and presence of ligands and/or binding molecules). Environment-induced conformational changes in IDPs can be detected by an NMR chemical shift [15,16]. This chemical shift, a sophisticated reporter of the backbone conformation, also indicates protein dynamics [17] and binding sites of ligands and/or other proteins. Compared with NMR of globular proteins, that of IDPs presents narrow line shapes [15,16,18–21] due to the highly dynamic nature of the polypeptide chain. Thanks to these narrow line shapes, the signal-to-noise ratio (S/N ratio) in NMR spectra of IDPs is usually high. However, S/N ratio can be low under physiological conditions (pH close to 7.0 and temperature about 30 ◦ C) due to the exchange of amide protons with the solvent. This limit can be mostly solved by using direct 13 C detection [22]. 3.2. EPR Spectroscopy EPR spectroscopy is useful for studying the structure and dynamics of macromolecules. Site-directed spin labeling (SDSL) EPR was developed by W. L Hubbel, and it is useful in characterizing protein structure and dynamics because most proteins do not have a paramagnetic center [23,24]. Nitroxides, which are stable free radicals, have an unpaired electron which is detectable by EPR. The EPR spectra of a nitroxide-labeled protein can provide information about: (i) the chain mobility at the label position; (ii) distance to other paramagnetic centers (using the double electron–electron resonance method); and (iii) accessibility of the solvent and oxygen [25]. Cysteine substitution mutagenesis of the protein, i.e., removing unwanted inherent cysteine (cysteine to serine) or introducing cysteines by site-directed mutagenesis, is the most common spin-labeling strategy [25]. The introduced thiol group is modified by spin-labeling agents such

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as (1-oxyl-2,2,5,5-tetramethyl-pyrroline-3-methyl)-methanethiosulfonate (MTSSL). Although MTSSL is small and has a minor impact on the protein structure [26], it is important to confirm differences in the structure and function of the spin-labeled protein compared with that of the wild-type. Several IDRs of IDPs were studied using the SDSL EPR technique [25,27–34]. The EPR spectra of IDRs showed sharp peaks due to the fast motion of the EPR probe, which results from the IDRs’ inherent flexibility. 3.3. CD Spectroscopy CD spectroscopy in the far ultraviolet region (175–250 nm) is a particularly useful and simple method for characterizing the secondary structure (alpha helix, beta structure, polyprolin type II (PPII) helix, and random coil) [35] of proteins because of the small amount of sample required, user-friendly method, and the possibility for sample recovery. Generally, CD spectra of IDPs present a strong negative band near 200 nm [18,21,36–38], similar to those of random coils, supporting the theory that IDPs lack well-defined secondary structures. CD spectra of IDRs dramatically change when a change in the secondary structure is induced because of environmental factors [39]. Because CD spectra of alpha helices and beta sheets have distinct properties (alpha helix: two minima at 208 and 222 nm and one maximum at 190 nm; beta sheet: one maximum at 198 nm and one minimum at approximately 217 nm), CD spectra provide quantitative structural data for IDPs. Although the distinct signature of PPII in CD spectrum, a positive peak at 217 nm, is sometimes obscured by negative contribution from alpha helices and beta sheets, the presence of PPII conformation in IDPs is shown by CD analysis [40]. 3.4. Single-Molecule Fluorescence Resonance Energy Transfer As described above, NMR, EPR, and CD spectroscopy can provide valuable information about structural features and/or dynamics of IDPs. However, these methods cannot detect the dynamic heterogeneity of each individual molecule because of ensemble and time averaging of the signal [15,41]. Single-molecule fluorescence techniques are good alternatives to overcome the limitations of the former methods. smFRET experiments, one of the single-molecule fluorescence methods, is useful for studying structural features of IDPs [27,41–47]. smFRET is based on the transfer of excited-state energy from the donor to acceptor fluorescence dyes. smFRET provides information relating to conformation and conformational transitions induced by environmental changes both in the individual molecule and at the subunit level [41]. smFRET also measures intra- or intermolecular distance between the donor and acceptor fluorophores. smFRET analysis requires two or more fluorescent dye labels at different positions on the protein. The most common approach for fluorescence labeling is cysteine substitution mutagenesis, as described above in the EPR experiment. Maleimide-conjugated chromophores can attach to cysteine in specific regions. Cyanine (Cy) [48] or Alexa Fluor series [49] are suitable dyes for smFRET because of their high fluorescence quantum yield, small size, and stability. Recently, cysteine labeling combined with methods that involve non-natural amino acids have been used for studying multiple regions using three-color FRET [47]. 4. Nopp140 as a Novel Class of IDP Nopp140 is a nucleolar protein and shuttles between the nucleolus and cytoplasm in mammalian cells [50]. It functions in nucleolus formation during cell division [51] and might be involved in the assembly of pre-ribosomal subunits [50]. It interacts with proteins essential for cellular function, such as RNA polymerase I [52], NAP57 [53], snRNPs [52], p80 coilin [50], and casein kinase 2 (CK2) [54]. Nopp140 contains 710 amino acids, and >80% of its structure is deemed to be disordered based on several disordered region prediction algorithms [27,55]. This prediction result together with CD spectra (Figure 2) and high protease sensitivity of Nopp140 [55] shows that Nopp140 is IDP.

Nopp140 can bind to both CK2 (catalytic and regulatory) subunits, and the binding affinity of the phosphorylated Nopp140 is higher than that of unphosphorylated Nopp140 [62,63]. Nopp140, particularly in its phosphorylated form, can also regulate the catalytic activity of CK2 [62]. These results suggest that Nopp140 is a CK2 substrate and is a negative regulator of CK2. A yeast twohybrid study using Nopp140 fragments showed that the C-terminus fragment (residues 528–704) Int. J. Mol. Sci. 2018, 19, 381 5 of 11 binds regions of the catalytic subunit (CK2α) [63].

Figure 2. CD spectra of: (A) Nopp140; (B) the N-terminal (residues 6–352); and (C) C-terminal Figure 2. CD spectra of: (A) Nopp140; (B) the N-terminal (residues 6–352); and (C) C-terminal fragments (residues 353–704) of Nopp140. Phosphorylated and unphosphorylated forms are fragments (residues 353–704) of Nopp140. Phosphorylated and unphosphorylated forms are represented as solid and dotted lines, respectively (experimental data taken from [18], with represented as solid and dotted lines, respectively (experimental data taken from [18], with permission permission KCS Publications). from ©2012from KCS©2012 Publications).

4.2. Biophysical Study of Nopp140 4.1. Interaction Between Nopp140 and CK2 To understand the CK2-regulating mechanism andphosphorylates conformationalvarious changeproteins of Nopp140 during CK2 is a ubiquitous serine/threonine kinase that related to itscrucial interaction with CK2, we prepared a variety of Nopp140 fragments based on the C-terminus cellular functions, such as cell division, proliferation, and signal transduction [56–59]. A high fragment of Nopp140. Surface plasmon resonance experiments using the fragments level of CK2 expression has been observed in many(SPR) cancers [60], suggesting thatNopp140 CK2 is a potential indicate that residues 568–596 and the phosphorylation of Ser574 in Nopp140 fragments are crucial anti-cancer drug target. for interaction CK2α (Figure 3)80[64]. Thesequences interaction between 568–596 regionbyand CK2α Nopp140with has approximately target that can bethe phosphorylated CK2 [61].is interrupted by D-myo-inositol 1,2,3,4,5,6-hexakisphosphate (IP 6 ) [64], an important regulator of many Nopp140 can bind to both CK2 (catalytic and regulatory) subunits, and the binding affinity of biological functions. These results together complex model structure of [62,63]. the 568–596 region the phosphorylated Nopp140 is higher thanwith thatthe of unphosphorylated Nopp140 Nopp140, and the CK2α suggests that the phosphorylated Ser574 and IPactivity 6 share of the same site for particularly in [27] its phosphorylated form, can also regulate the catalytic CK2 [62].binding These results CK2 [64], which is the key site for regulation of CK2 activity by Nopp140. suggest that Nopp140 is a CK2 substrate and is a negative regulator of CK2. A yeast two-hybrid study using Nopp140 fragments showed that the C-terminus fragment (residues 528–704) binds regions of the catalytic subunit (CK2α) [63]. 4.2. Biophysical Study of Nopp140 To understand the CK2-regulating mechanism and conformational change of Nopp140 during its interaction with CK2, we prepared a variety of Nopp140 fragments based on the C-terminus fragment of Nopp140. Surface plasmon resonance (SPR) experiments using the Nopp140 fragments indicate that residues 568–596 and the phosphorylation of Ser574 in Nopp140 fragments are crucial for interaction with CK2α (Figure 3) [64]. The interaction between the 568–596 region and CK2α is interrupted Figure 3. SPR analysis of the interaction between of CK2 (CK2α) Nopp140 by D-myo-inositol 1,2,3,4,5,6-hexakisphosphate (IP6 )catalytic [64], an subunit important regulator of and many biological fragments. “p” results indicates the phosphorylation of Ser574 in Nopp140 fragments. (A) Binding affinities functions. These together with the complex model structure of the 568–596 region and the of [27] Nopp140 fragments CK2α. SPR sensorgrams (B) Nopp140 andfor(C) the[64], CK2α suggests that thewith phosphorylated Ser574 and IPof: the same(568–596); binding site CK2 6 share phosphorylation Ser574 in Nopp140 CK2α. These sensorgrams were obtained for which is the key siteoffor regulation of CK2(568–596) activity with by Nopp140. 0.1 (orange), 1 (green), (purple), 100 (blue), and(MTSSL) 1000 μM Nopp140 (red) of Nopp140 fragments, respectively EPR spectra created10 using the spin-labeled fragments showed sharp peaks (experimental takenoffrom [64], with group permission from ©2013 National of Sciences). because of the fastdata motion the nitroxide in MTSSL (Figure 4) [27].Academy Among the MTSSL-labeled Nopp140s, only the EPR spectrum of the 589 fragments (those spin-labeled at residue 589) was changed spectra created using the spin-labeled (MTSSL) Nopp140 fragments sharp peaks to a EPR broader line shape in the presence of CK2α (Figure 4) [27]. These results imply showed that MTSSL-labeled because of the fastlabeled motion the nitroxide group MTSSL 4) [27]. Among the MTSSL-labeled Nopp140s were inof disordered regions andinthe region(Figure near amino acid 589 might be crucial for Nopp140s, only the EPR spectrum of the 589 fragments (those spin-labeled at residue 589) was binding to CK2. 1 15 changed a broader line shapesingle in thequantum presencecoherence of CK2α (Figure 4) [27]. These results imply that 2D toHN heteronuclear spectroscopy (HSQC) NMR spectra of the C-terminus fragment presented a narrow line shape (Figure 5), typical of the 1 H-15 N HSQC NMR spectra of IDPs [18], which is another result that supports the theory that the C-terminus of Nopp140 is a disordered region. NMR spectra of Nopp140 are shifted when C-terminus fragments are mixed with mitoxantrone (Figure 5), an anti-cancer agent, demonstrating that Nopp140 interacts

permission from ©2012 KCS Publications).

4.2. Biophysical Study of Nopp140 To understand the CK2-regulating mechanism and conformational change of Nopp140 during its interaction with CK2, we prepared a variety of Nopp140 fragments based on the C-terminus Int. J. Mol. Sci. 2018, 19, 381 6 of 11 fragment of Nopp140. Surface plasmon resonance (SPR) experiments using the Nopp140 fragments indicate that residues 568–596 and the phosphorylation of Ser574 in Nopp140 fragments are crucial with mitoxantrone An (Figure in vitro 3) kinase of CK2 showed thatthe the568–596 reducedregion inhibitory for interaction with[18]. CK2α [64]. assay The interaction between and activity CK2α is of Nopp140 against CK2 by IP was recovered by mitoxantrone [18]. Results of the kinase 6 interrupted by D-myo-inositol 1,2,3,4,5,6-hexakisphosphate (IP6) [64], an important regulator ofassay, many 1 15 together with the H- These N HSQC NMR experiment of Nopp140 the presence ofof mitoxantrone, biological functions. results together with the complex in model structure the 568–596 imply region that regulation of CK2 by Nopp140 by enhancing thesame interaction between andmitoxantrone the CK2α [27]stabilizes suggeststhe that the phosphorylated Ser574 and IP6 share the binding site for Nopp140 and CK2 [18]. CK2 [64], which is the key site for regulation of CK2 activity by Nopp140.

Figure 3. SPR analysis of the interaction between catalytic subunit of CK2 (CK2α) and Nopp140 Figure 3. SPR analysis of the interaction between catalytic subunit of CK2 (CK2α) and fragments. “p” indicates the phosphorylation of Ser574 in Nopp140 fragments. (A) Binding affinities Int. J. Mol. Sci. 2018, 0, x FOR PEER REVIEW the phosphorylation of Ser574 in Nopp140 fragments. (A) Binding6 of 10 Nopp140 fragments. “p” indicates of Nopp140 fragments with CK2α. SPR sensorgrams of: (B) Nopp140 (568–596); and (C) the affinities of Nopp140 fragments with CK2α. SPR sensorgrams of: (B) Nopp140 (568–596); and (C) the phosphorylation of Ser574 in Nopp140 (568–596) with regions CK2α. These sensorgrams were amino obtained for 589 MTSSL-labeled Nopp140s were labeled (568–596) in disordered andsensorgrams the region were near phosphorylation of Ser574 in Nopp140 with CK2α. These obtainedacid for 0.1 (orange), 1 (green), 10 to (purple), 100 (blue), and 1000 μM (red) of Nopp140 fragments, respectively might0.1 be(orange), crucial 1for binding CK2. 100 (green), 10 (purple), (blue), and 1000 µM (red) of Nopp140 fragments, respectively (experimentaldata datataken takenfrom from[64], [64],with withpermission permissionfrom from©2013 ©2013National NationalAcademy AcademyofofSciences). Sciences). (experimental

EPR spectra created using the spin-labeled (MTSSL) Nopp140 fragments showed sharp peaks because of the fast motion of the nitroxide group in MTSSL (Figure 4) [27]. Among the MTSSL-labeled Nopp140s, only the EPR spectrum of the 589 fragments (those spin-labeled at residue 589) was changed to a broader line shape in the presence of CK2α (Figure 4) [27]. These results imply that

Figure spectra of spin-labeled C-terminus Nopp140 fragments. The EPRThe spectra Nopp140 Figure 4. 4. EPR EPR spectra of spin-labeled C-terminus Nopp140 fragments. EPRofspectra of fragments in the absence and the presence of CK2α are represented are shown as solid (black) and Nopp140 fragments in the absence and the presence of CK2α are represented are shown as solid dotted-lines (red), respectively (experimental data takendata from [27],from with permission from ©2016 (black) and dotted-lines (red), respectively (experimental taken [27], with permission from Elsevier). ©2016 Elsevier).

2D 1H-15N heteronuclear single quantum coherence spectroscopy (HSQC) NMR spectra of the Double fluorescence-labeled (Cy3 and Cy5 probes) Nopp140 fragments were used for FRET C-terminus fragment presented a narrow line shape (Figure 5), typical of the 1H-15N HSQC NMR experiments [27]. An ensemble FRET experiment showed that the FRET intensity of the Nopp140-352/589 spectra of IDPs [18], which is another result that supports the theory that the C-terminus of Nopp140 fragment was higher than that of other fragments (in order, from high to low: Nopp140-352/589 > is a disordered region. NMR spectra of Nopp140 are shifted when C-terminus fragments are mixed Nopp140-352/467 > Nopp140-352/660 > Nopp140-352/704) [27], indicating that the estimated distances with mitoxantrone (Figure 5), an anti-cancer agent, demonstrating that Nopp140 interacts with between the fluorescence labels in solution are not proportional to the number of amino acids. This mitoxantrone [18]. An in vitro kinase assay of CK2 showed that the reduced inhibitory activity of result implies that the structure of Nopp140 is not linear or in a locally constrained conformation. Nopp140 against CK2 by IP6 was recovered by mitoxantrone [18]. Results of the kinase assay, together The Nopp140-352/589 intensity substantially decreased in the presence of CK2α, which suggests that with the 1H-15N HSQC NMR experiment of Nopp140 in the presence of mitoxantrone, imply that conformational distribution or the structure of the region near amino acid 589 is changed when CK2α mitoxantrone stabilizes the regulation of CK2 by Nopp140 by enhancing the interaction between binds to the C-terminus of Nopp140 [27]. Single-molecule FRET experiments using double-labeled Nopp140 and CK2 [18]. Nopp140-352/589 fragments provided more detailed information (Figure 6). The FRET histogram of Nopp140-352/589 showed two Gaussian distributions (the low- and middle-FRET population) (Figure 6A) [27]. Because the FRET histogram in the presence of 6 M guanidine hydrochloride presented only one Gaussian distribution (Figure 6C) [27], the C-terminus fragment of Nopp140 has at least two structural conformations in its isolated form. Consistent with the ensemble FRET results, approximately 30% of the middle-FRET population shifted to low FRET in the presence of CK2α (Figure 6B) [27]. This population change indicated that the structure of Nopp140 became more restricted in its conformation because of its interaction with CK2α.

is a disordered region. NMR spectra of Nopp140 are shifted when C-terminus fragments are mixed with mitoxantrone (Figure 5), an anti-cancer agent, demonstrating that Nopp140 interacts with mitoxantrone [18]. An in vitro kinase assay of CK2 showed that the reduced inhibitory activity of Nopp140 against CK2 by IP6 was recovered by mitoxantrone [18]. Results of the kinase assay, together 1H-15N HSQC NMR experiment of Nopp140 in the presence of mitoxantrone, imply that with the Sci. Int. J. Mol. 2018, 19, 381 7 of 11 mitoxantrone stabilizes the regulation of CK2 by Nopp140 by enhancing the interaction between Nopp140 and CK2 [18].

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estimated distances between the fluorescence labels in solution are not proportional to the number of amino acids. This result implies that the structure of Nopp140 is not linear or in a locally constrained conformation. The Nopp140-352/589 intensity substantially decreased in the presence of CK2α, which suggests that conformational distribution or the structure of the region near amino acid 589 is changed when CK2α binds to the C-terminus of Nopp140 [27]. Single-molecule FRET experiments using double-labeled Nopp140-352/589 fragments provided more detailed information (Figure 6). The FRET histogram of Nopp140-352/589 showed two Gaussian distributions (the lowand middle-FRET population) (Figure 6A) [27]. Because the FRET histogram in the presence of 6 M guanidine hydrochloride presented only one Gaussian distribution (Figure 6C) [27], the C-terminus fragment of Nopp140 has at least two structural conformations in its isolated form. Consistent with the ensemble FRET results, approximately 30% of the middle-FRET population shifted to low FRET Figure 5. 11H-1515N spectra of the C-terminus Nopp140 fragment (residues 528–704, black) and with Figure 5. H-ofNCK2α spectra(Figure of the C-terminus Nopp140 fragment (residues 528–704,that black) with in the presence 6B)data [27]. taken This population change indicated theand structure of mitoxantrone (red) (experimental from [18], with permission from ©2012 KCS mitoxantrone (red) (experimental data taken from [18], with permission from ©2012 KCS Publications). Nopp140 became more restricted in its conformation because of its interaction with CK2α. Publications). Double fluorescence-labeled (Cy3 and Cy5 probes) Nopp140 fragments were used for FRET experiments [27]. An ensemble FRET experiment showed that the FRET intensity of the Nopp140-352/589 fragment was higher than that of other fragments (in order, from high to low: Nopp140-352/589 > Nopp140-352/467 > Nopp140-352/660 > Nopp140-352/704) [27], indicating that the

Figure 6. smFRET histograms of double-labeled: (A) Nopp140-352/589 only; (B) with CK2α; and Figure 6. smFRET histograms of double-labeled: (A) Nopp140-352/589 only; (B) with CK2α; and (C) in 6 M guanidine hydrochloride. Two Gaussian peaks distributions having a FRET-peak centered (C) in 6 M guanidine hydrochloride. Two Gaussian peaks distributions having a FRET-peak centered at 0.03 and 0.45 E are shown as blue and red, respectively (experimental data taken from [27], with at 0.03 and 0.45 E are shown as blue and red, respectively (experimental data taken from [27], permission from ©2016 Elsevier). with permission from ©2016 Elsevier).

5. Conclusion 5. Conclusions IDPs harboring a defined disordered region or IDPs mostly comprising disordered regions are IDPs harboring a defined disordered region or IDPs mostly comprising disordered regions are commonly observed in most organisms, and their significance in various biological phenomena is commonly observed in most organisms, and their significance in various biological phenomena is clear. In particular, IDPs in which the disordered regions cover >60–80% of the whole protein are of clear. In particular, IDPs in which the disordered regions cover >60–80% of the whole protein are of interest with respect to their mechanism of action. These IDPs possess several motifs which can serve interest with respect to their mechanism of action. These IDPs possess several motifs which can serve as interaction sites for different binding proteins, and the activity of a binding protein can be modified as interaction sites for different binding proteins, and the activity of a binding protein can be modified upon interaction with IDPs. Nopp140 is a good example of such IDP. It lacks any well-folded domains upon interaction with IDPs. Nopp140 is a good example of such IDP. It lacks any well-folded domains in its entire sequence of 710 aa, and it can interact with various proteins or RNAs, such as CK2α, RNA in its entire sequence of 710 aa, and it can interact with various proteins or RNAs, such as CK2α, polymerase I, NAP57, p80 coilin, and snRNPs. The modulation of the kinase activity of CK2 by RNA polymerase I, NAP57, p80 coilin, and snRNPs. The modulation of the kinase activity of CK2 by Nopp140 has been studied in detail. CK2-dependent phosphorylation of Nopp140 and inhibition of Nopp140 has been studied in detail. CK2-dependent phosphorylation of Nopp140 and inhibition the kinase activity of CK2 by phosphorylated Nopp140 indicates that Nopp140 plays the role of a of the kinase activity of CK2 by phosphorylated Nopp140 indicates that Nopp140 plays the role of negative regulator to CK2 [62,64]. Further biophysical studies on Nopp140 during its interaction with a negative regulator to CK2 [62,64]. Further biophysical studies on Nopp140 during its interaction CK2 by smFRET, EPR, and NMR revealed increased rigid conformation of Nopp140 at or near the with CK2 by smFRET, EPR, and NMR revealed increased rigid conformation of Nopp140 at or near CK2 binding site [27]. It could be said that the interacting molecules of Nopp140 are specifically positioned. A comprehensive study of the dynamic interaction between proteins (or RNAs) mediated by mostly disordered proteins will reveal much that is waiting to be uncovered. Acknowledgments: This work was supported by grants from the Bio & Medical Technology Development Program (2017M3A9C8060552 and 2017M3A9C8060560) through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning. This work was also supported by a grant

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the CK2 binding site [27]. It could be said that the interacting molecules of Nopp140 are specifically positioned. A comprehensive study of the dynamic interaction between proteins (or RNAs) mediated by mostly disordered proteins will reveal much that is waiting to be uncovered. Acknowledgments: This work was supported by grants from the Bio & Medical Technology Development Program (2017M3A9C8060552 and 2017M3A9C8060560) through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning. This work was also supported by a grant from the Mid-Career Research Program (2016R1A2B4009952) of National Research Foundation of Korea. Author Contributions: Jung-Hyun Na and Yeon Gyu Yu prepared the manuscript. Won-Kyu Lee checked the references and made figures of manuscript. Yeon Gyu Yu provided critical feedback and contributed to the final version of the manuscript. Conflicts of Interest: The authors declare no competing financial interests.

References 1.

2. 3. 4. 5. 6.

7. 8.

9.

10.

11. 12.

13. 14. 15. 16. 17.

Van der Lee, R.; Buljan, M.; Lang, B.; Weatheritt, R.J.; Daughdrill, G.W.; Dunker, A.K.; Fuxreiter, M.; Gough, J.; Gsponer, J.; Jones, D.T.; et al. Classification of Intrinsically Disordered Regions and Proteins. Chem. Rev. 2014, 114, 6589–6631. [CrossRef] [PubMed] Dunker, A.K.; Obradovic, Z.; Romero, P.; Garner, E.C.; Brown, C.J. Intrinsic protein disorder in complete genomes. Genome inform. Workshop Genome Inform. 2000, 11, 161–171. Wang Jabs, E.; Li, X.; Coss, C.A.; Taylor, E.W.; Meyers, D.A.; Weber, J.L. Mapping the Treacher Collins syndrome locus to 5q31.3→q33.3. Genomics 1991, 11, 193–198. [CrossRef] Oldfield, C.J.; Dunker, A.K. Intrinsically disordered proteins and intrinsically disordered protein regions. Ann. Rev. Biochem. 2014, 83, 553–584. [CrossRef] [PubMed] Dosztanyi, Z.; Meszaros, B.; Simon, I. Bioinformatical approaches to characterize intrinsically disordered/unstructured proteins. Brief. Bioinform. 2010, 11, 225–243. [CrossRef] [PubMed] Weinreb, P.H.; Zhen, W.; Poon, A.W.; Conway, K.A.; Lansbury, P.T. NACP, A Protein Implicated in Alzheimer’s Disease and Learning, Is Natively Unfolded. Biochemistry 1996, 35, 13709–13715. [CrossRef] [PubMed] Dyson, H.J.; Wright, P.E. Intrinsically unstructured proteins and their functions. Nat. Rev. Mol. Cell Biol. 2005, 6, 197–208. [CrossRef] [PubMed] Kumar, D.; Sharma, N.; Giri, R. Therapeutic Interventions of Cancers Using Intrinsically Disordered Proteins as Drug Targets: c-Myc as Model System. Cancer Informatics 2017, 16, 1176935117699408. [CrossRef] [PubMed] Dosztanyi, Z.; Csizmok, V.; Tompa, P.; Simon, I. IUPred: web server for the prediction of intrinsically unstructured regions of proteins based on estimated energy content. Bioinformatics 2005, 21, 3433–3434. [CrossRef] [PubMed] Dunker, A.K.; Lawson, J.D.; Brown, C.J.; Williams, R.M.; Romero, P.; Oh, J.S.; Oldfield, C.J.; Campen, A.M.; Ratliff, C.M.; Hipps, K.W.; et al. Intrinsically disordered protein. J. Mol. Graph. Model. 2001, 19, 26–59. [CrossRef] Moll, U.M.; Petrenko, O. The MDM2-p53 interaction. Mol. Cancer Res. 2003, 1, 1001–1008. [PubMed] Kussie, P.H.; Gorina, S.; Marechal, V.; Elenbaas, B.; Moreau, J.; Levine, A.J.; Pavletich, N.P. Structure of the MDM2 oncoprotein bound to the p53 tumor suppressor transactivation domain. Science 1996, 274, 948–953. [CrossRef] [PubMed] Pomeranz Krummel, D.A.; Oubridge, C.; Leung, A.K.W.; Li, J.; Nagai, K. Crystal structure of human spliceosomal U1 snRNP at 5.5 Å resolution. Nature 2009, 458, 475–480. [CrossRef] [PubMed] Eliezer, D. Biophysical characterization of intrinsically disordered proteins. Curr. Opin. Struct. Biol. 2009, 19, 23–30. [CrossRef] [PubMed] Konrat, R. NMR contributions to structural dynamics studies of intrinsically disordered proteins. J. Magn. Reson. 2014, 241, 74–85. [CrossRef] [PubMed] Gibbs, E.B.; Cook, E.C.; Showalter, S.A. Application of NMR to studies of intrinsically disordered proteins. Arch. Biochem. Biophys. 2017, 628, 57–70. [CrossRef] [PubMed] Berjanskii, M.; Wishart, D.S. NMR: prediction of protein flexibility. Nat. Protoc. 2006, 1, 683–688. [CrossRef] [PubMed]

Int. J. Mol. Sci. 2018, 19, 381

18.

19. 20. 21.

22.

23. 24. 25. 26.

27.

28.

29.

30.

31.

32.

33.

34.

35. 36.

9 of 11

Lee, W.K.; Lee, S.Y.; Na, J.H.; Jang, S.; Park, C.R.; Kim, S.Y.; Lee, S.H.; Han, K.; Yu, Y.G. Mitoxantrone Binds to Nopp140, an Intrinsically Unstructured Protein, and Modulate its Interaction with Protein Kinase CK2. Bull Korean Chem. Soc. 2012, 33, 2005–2011. [CrossRef] Kim, D.H.; Lee, C.; Lee, S.H.; Kim, K.T.; Han, J.J.; Cha, E.J.; Lim, J.E.; Cho, Y.J.; Hong, S.H.; Han, K.H. The Mechanism of p53 Rescue by SUSP4. Angew. Chem. 2017, 56, 1278–1282. [CrossRef] [PubMed] Kim, D.H.; Wright, A.; Han, K.H. An NMR study on the intrinsically disordered core transactivation domain of human glucocorticoid receptor. BMB Rep. 2017, 50, 522–527. [CrossRef] [PubMed] Arai, M.; Sugase, K.; Dyson, H.J.; Wright, P.E. Conformational propensities of intrinsically disordered proteins influence the mechanism of binding and folding. Proc. Natl. Acad. Sci. USA 2015, 112, 9614–9619. [CrossRef] [PubMed] Bermel, W.; Bertini, I.; Chill, J.; Felli, I.C.; Haba, N.; Kumar, M.V.V.; Pierattelli, R. Exclusively heteronuclear (13)C-detected amino-acid-selective NMR experiments for the study of intrinsically disordered proteins (IDPs). ChemBioChem 2012, 13, 2425–2432. [CrossRef] [PubMed] Hubbell, W.L.; Altenbach, C. Investigation of structure and dynamics in membrane proteins using site-directed spin labeling. Curr. Opin. Struct. Biol. 1994, 4, 566–573. [CrossRef] Hubbell, W.L.; Gross, A.; Langen, R.; Lietzow, M.A. Recent advances in site-directed spin labeling of proteins. Curr. Opin. Struct. Biol. 1998, 8, 649–656. [CrossRef] Drescher, M. EPR in protein science : Intrinsically disordered proteins. Top. Curr. Chem. 2012, 321, 91–119. [PubMed] McHaourab, H.S.; Lietzow, M.A.; Hideg, K.; Hubbell, W.L. Motion of Spin-Labeled Side Chains in T4 Lysozyme. Correlation with Protein Structure and Dynamics. Biochemistry 1996, 35, 7692–7704. [CrossRef] [PubMed] Na, J.H.; Lee, W.K.; Kim, Y.; Jeong, C.; Song, S.S.; Cha, S.S.; Han, K.H.; Shin, Y.K.; Yu, Y.G. Biophysical characterization of the structural change of Nopp140, an intrinsically disordered protein, in the interaction with CK2α. Biochem. Biophys. Res. Commun. 2016, 477, 181–187. [CrossRef] [PubMed] Morin, B.; Bourhis, J.-M.; Belle, V.; Woudstra, M.; Carrière, F.; Guigliarelli, B.; Fournel, A.; Longhi, S. Assessing induced folding of an intrinsically disordered protein by site-directed spin-labeling electron paramagnetic resonance spectroscopy. J. Phys. Chem. B 2006, 110, 20596–20608. [CrossRef] [PubMed] Belle, V.; Rouger, S.; Costanzo, S.; Liquiere, E.; Strancar, J.; Guigliarelli, B.; Fournel, A.; Longhi, S. Mapping alpha-helical induced folding within the intrinsically disordered C-terminal domain of the measles virus nucleoprotein by site-directed spin-labeling EPR spectroscopy. Proteins 2008, 73, 973–988. [CrossRef] [PubMed] Török, M.; Milton, S.; Kayed, R.; Wu, P.; McIntire, T.; Glabe, C.G.; Langen, R. Structural and Dynamic Features of Alzheimer’s Aβ Peptide in Amyloid Fibrils Studied by Site-directed Spin Labeling. J. Biol. Chem. 2002, 277, 40810–40815. [CrossRef] [PubMed] Murakami, K.; Hara, H.; Masuda, Y.; Ohigashi, H.; Irie, K. Distance measurement between Tyr10 and Met35 in amyloid beta by site-directed spin-labeling ESR spectroscopy: Implications for the stronger neurotoxicity of Abeta42 than Abeta40. Chembiochem 2007, 8, 2308–2314. [CrossRef] [PubMed] Ionut Iurascu, M.; Cozma, C.; Tomczyk, N.; Rontree, J.; Desor, M.; Drescher, M.; Przybylski, M. Structural characterization of beta-amyloid oligomer-aggregates by ion mobility mass spectrometry and electron spin resonance spectroscopy. Anal. Bioanal. Chem. 2009, 395, 2509–2519. [CrossRef] [PubMed] Sepkhanova, I.; Drescher, M.; Meeuwenoord, N.J.; Limpens, R.W.; Koning, R.I.; Filippov, D.V.; Huber, M. Monitoring Alzheimer Amyloid Peptide Aggregation by EPR. Appl. Magn. Reson. 2009, 36, 209–222. [CrossRef] [PubMed] Drescher, M.; Godschalk, F.; Veldhuis, G.; van Rooijen, B.D.; Subramaniam, V.; Huber, M. Spin-label EPR on alpha-synuclein reveals differences in the membrane binding affinity of the two antiparallel helices. Chembiochem 2008, 9, 2411–2416. [CrossRef] [PubMed] Johnson, W.C. Jr. Protein secondary structure and circular dichroism: a practical guide. Proteins 1990, 7, 205–214. [CrossRef] [PubMed] Maestro, B.; Galán, B.; Alfonso, C.; Rivas, G.; Prieto, M.A.; Sanz, J.M. A New Family of Intrinsically Disordered Proteins: Structural Characterization of the Major Phasin PhaF from Pseudomonas putida KT2440. PLoS ONE 2013, 8, e56904. [CrossRef] [PubMed]

Int. J. Mol. Sci. 2018, 19, 381

10 of 11

37.

Balu, R.; Knott, R.; Cowieson, N.P.; Elvin, C.M.; Hill, A.J.; Choudhury, N.R.; Dutta, N.K. Structural ensembles reveal intrinsic disorder for the multi-stimuli responsive bio-mimetic protein Rec1-resilin. Sci. Rep. 2015, 5, 10896. [CrossRef] [PubMed] 38. Hamdi, K.; Salladini, E.; O’Brien, D.P.; Brier, S.; Chenal, A.; Yacoubi, I.; Longhi, S. Structural disorder and induced folding within two cereal, ABA stress and ripening (ASR) proteins. Sci. Rep. 2017, 7, 15544. [CrossRef] [PubMed] 39. Chemes, L.B.; Alonso, L.G.; Noval, M.G.; de Prat-Gay, G. Circular dichroism techniques for the analysis of intrinsically disordered proteins and domains. In Intrinsically Disordered Protein Analysis: Volume 1, Methods and Experimental Tools; Uversky, V.N., Dunker, A.K., Eds.; Humana Press: Totowa, NJ, USA, 2012; pp. 387–404. 40. Tompa, P.; Fersht, A. Structure and Function of Intrinsically Disordered Proteins; CRC Press: Boca Raton, FL, USA, 2009; pp. 126–127. 41. Brucale, M.; Schuler, B.; Samorì, B. Single-Molecule Studies of Intrinsically Disordered Proteins. Chem. Rev. 2014, 114, 3281–3317. [CrossRef] [PubMed] 42. Gambin, Y.; VanDelinder, V.; Ferreon, A.C.M.; Lemke, E.A.; Groisman, A.; Deniz, A.A. Visualizing a one-way protein encounter complex by ultrafast single-molecule mixing. Nat. Methods 2011, 8, 239. [CrossRef] [PubMed] 43. Lamboy, J.A.; Kim, H.; Dembinski, H.; Ha, T.; Komives, E.A. Single-molecule FRET reveals the native-state dynamics of the IkappaBalpha ankyrin repeat domain. J. Mol. Biol. 2013, 425, 2578–2590. [CrossRef] [PubMed] 44. Ferreon, A.C.M.; Gambin, Y.; Lemke, E.A.; Deniz, A.A. Interplay of α-synuclein binding and conformational switching probed by single-molecule fluorescence. Proc. Natl. Acad. Sci. USA 2009, 106, 5645–5650. [CrossRef] [PubMed] 45. Mukhopadhyay, S.; Krishnan, R.; Lemke, E.A.; Lindquist, S.; Deniz, A.A. A natively unfolded yeast prion monomer adopts an ensemble of collapsed and rapidly fluctuating structures. Proc. Natl. Acad. Sci. USA 2007, 104, 2649–2654. [CrossRef] [PubMed] 46. Lee, T.; Moran-Gutierrez, C.R.; Deniz, A.A. Probing protein disorder and complexity at single-molecule Int. J. Mol. Sci. 2018, 0,Semin. x FOR Cell PEERDev. REVIEW 10 of 10 resolution. Biol. 2015, 37 (Supplement C), 26–34. [CrossRef] [PubMed] 47. Milles, S.; Koehler, C.; Gambin, Y.; Deniz, A.A.; Lemke, E.A. Intramolecular three-colour single pair FRET of 47. Milles, S.; Koehler, C.; Gambin, Y.;with Deniz, A.A.; Lemke, E.A. Intramolecular three-colour single pair FRET intrinsically disordered proteins increased dynamic range. Mol. Biosyst. 2012, 8, 2531–2534. [CrossRef] of [PubMed] intrinsically disordered proteins with increased dynamic range. Mol. Biosyst. 2012, 8, 2531–2534. 48.48. Mujumdar, R.B.; Ernst, L.A.; Mujumdar, S.R.; Lewis, C.J.; Waggoner, A.S. Cyanine dye labeling reagents: Mujumdar, R.B.; Ernst, L.A.; Mujumdar, S.R.; Lewis, C.J.; Waggoner, A.S. Cyanine dye labeling reagents: sulfoindocyanine succinimidyl esters. Bioconjug. Chem. 1993, 4, 105–111. sulfoindocyanine succinimidyl esters. Bioconjug. Chem. 1993, 4, 105–111. [CrossRef] [PubMed] 49.49. Panchuk-Voloshina, J.; Bhalgat, Bhalgat,M.K.; M.K.;Millard, Millard,P.J.; P.J.; Mao, Leung, Panchuk-Voloshina,N.; N.;Haugland, Haugland,R.P.; R.P.; Bishop-Stewart, J.; Mao, F.; F.; Leung, W.Y.; W.Y.; Haugland, R.P. Alexa a of series new fluorescent yield exceptionally bright, Haugland, R.P. Alexa dyes, dyes, a series new of fluorescent dyes thatdyes yieldthat exceptionally bright, photostable photostable J. Histochem. 1999, 47, 1179–1188. conjugates.conjugates. J. Histochem. Cytochem. Cytochem. 1999, 47, 1179–1188. [CrossRef] [PubMed] 50.50. Isaac, C.;C.; Yang, Molecular Link LinkBetween Betweenthe theNucleolus Nucleolusand andthe Isaac, Yang,Y.;Y.;Thomas ThomasMeier, Meier,U. U.Nopp140 Nopp140 Functions Functions as a Molecular theCoiled CoiledBodies. Bodies.J.J.Cell CellBiol. Biol.1998, 1998,142, 142,319–329. 319–329.[CrossRef] [PubMed] 51.51. Meier, U.T.; Blobel, G.G. AA nuclear localization signal binding protein in in thethe nucleolus. J. Cell Biol. 1990, 111, Meier, U.T.; Blobel, nuclear localization signal binding protein nucleolus. J. Cell Biol. 1990, 111, 2235–2245. 2235–2245. [CrossRef] [PubMed] 52.52. Yang, Y.;Y.;Isaac, V.;Meier, Meier,U.T. U.T.Conserved Conservedcomposition composition mammalian Yang, Isaac,C.; C.;Wang, Wang,C.; C.;Dragon, Dragon,F.; F.;Poga Pogac̆ić,,V.; ofof mammalian box box H/ACA and C/D small nucleolar ribonucleoprotein particles and interaction their interaction with the H/ACA and boxbox C/D small nucleolar ribonucleoprotein particles and their with the common common factor Nopp140. Mol. Biol. Cell11, 2000, 11, 567–577. factor Nopp140. Mol. Biol. Cell 2000, 567–577. [CrossRef] [PubMed] Meier,U.T.; U.T.;Blobel, Blobel, G., G. NAP57, nucleolar protein with a putative homolog in yeast 53.53. Meier, NAP57,aamammalian mammalian nucleolar protein with a putative homolog in and yeastbacteria. and J. Cell Biol. 1994, 1505–1514. [CrossRef] [PubMed] bacteria. J. Cell Biol.127, 1994, 127, 1505–1514. Meier, U.T.; Dobrowolska, Krebs, E.G. Specific Interaction between Casein Kinase 2 and 54.54. Li,Li, D.;D.; Meier, U.T.; Dobrowolska, G.;G.; Krebs, E.G. Specific Interaction between Casein Kinase 2 and thethe Nucleolar Protein Nopp140. J. Biol. Chem. 1997, 272, 3773–3779. [CrossRef] [PubMed] Nucleolar Protein Nopp140. J. Biol. Chem. 1997, 272, 3773–3779. Kim, Y.K.; Lee, W.K.; Jin, Lee, K.J.; Jeon, Yu, Y.G.Doxorubicin Doxorubicin binds un-phosphorylated form 55.55. Kim, Y.K.; Lee, W.K.; Jin, Y.;Y.; Lee, K.J.; Jeon, H.;H.; Yu, Y.G. binds toto un-phosphorylated form of of hNopp140 and reduces protein kinase CK2-dependent phosphorylation hNopp140. J. Biochem. Mol. Biol. hNopp140 and reduces protein kinase CK2-dependent phosphorylation ofof hNopp140. J. Biochem. Mol. Biol. 2006, 774–781. [CrossRef] [PubMed] 2006, 39,39, 774–781. Henriksson, Lüscher, Proteins Myc network: essential regulators growthand and 56.56. Henriksson, M.;M.; Lüscher, B. B. Proteins of of thethe Myc Network: Essential Regulators of of Cellcell Growth differentiation. Adv. Cancer Res. 1996, 68, 109–182. Differentiation. In Adv. Cancer Res., Vande Woude, G. F.; Klein, G., Eds. Academic Press: Salt Lake City,

57.

58.

UT, USA, 1996; Volume 68, pp. 109–182. Keller, D.M.; Zeng, X.; Wang, Y.; Zhang, Q.H.; Kapoor, M.; Shu, H.; Goodman, R.; Lozano, G.; Zhao, Y.; Lu, H. A DNA damage–induced p53 serine 392 kinase complex contains CK2, hSpt16, and SSRP1. Mol. Cell 2001, 7, 283–292. Kulartz, M.; Kreitz, S.; Hiller, E.; Damoc, E.-C.; Przybylski, M.; Knippers, R. Expression and phosphorylation of the replication regulator protein geminin. Biochem. Biophys. Res. Commun. 2003, 305, 412–420.

Int. J. Mol. Sci. 2018, 19, 381

57.

58. 59. 60. 61. 62.

63.

64.

11 of 11

Keller, D.M.; Zeng, X.; Wang, Y.; Zhang, Q.H.; Kapoor, M.; Shu, H.; Goodman, R.; Lozano, G.; Zhao, Y.; Lu, H. A DNA damage–induced p53 serine 392 kinase complex contains CK2, hSpt16, and SSRP1. Mol. Cell 2001, 7, 283–292. [CrossRef] Kulartz, M.; Kreitz, S.; Hiller, E.; Damoc, E.-C.; Przybylski, M.; Knippers, R. Expression and phosphorylation of the replication regulator protein geminin. Biochem. Biophys. Res. Commun. 2003, 305, 412–420. [CrossRef] Lin, A.; Frost, J.; Deng, T.; Smeal, T.; Al-Alawi, N.; Kikkawa, U.; Hunter, T.; Brenner, D.; Karin, M. Casein kinase II is a negative regulator of c-Jun DNA binding and AP-1 activity. Cell 1992, 70, 777–789. [CrossRef] Tawfic, S.; Yu, S.; Wang, H.; Faust, R.; Davis, A.; Ahmed, K. Protein kinase CK2 signal in neoplasia. Histol. Histopathol. 2001, 16, 573–582. [PubMed] Pai, C.Y.; Chen, H.K.; Sheu, H.L.; Yeh, N.H. Cell-cycle-dependent alterations of a highly phosphorylated nucleolar protein p130 are associated with nucleologenesis. J. Cell Sci. 1995, 108, 1911–1920. [PubMed] Kim, Y.-K.; Lee, K.J.; Jeon, H.; Yu, Y.G. Protein kinase CK2 is inhibited by human nucleolar phosphoprotein p140 in an inositol hexakisphosphate-dependent manner. J. Biol. Chem. 2006, 281, 36752–36757. [CrossRef] [PubMed] Lee, W.K.; Lee, S.Y.; Kim, W.I.; Rho, Y.H.; Bae, Y.S.; Lee, C.; Kim, I.Y.; Yu, Y.G. Characterization of the InsP6-dependent interaction between CK2 and Nopp140. Biochem. Biophys. Res. Commun. 2008, 376, 439–444. [CrossRef] [PubMed] Lee, W.K.; Son, S.H.; Jin, B.S.; Na, J.H.; Kim, S.Y.; Kim, K.H.; Kim, E.E.; Yu, Y.G.; Lee, H.H. Structural and functional insights into the regulation mechanism of CK2 by IP6 and the intrinsically disordered protein Nopp140. Proc. Natl. Acad. Sci. USA 2013, 110, 19360–19365. [CrossRef] [PubMed] © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).